High number of SARS-CoV-2 persistent infections uncovered through genetic analysis of samples from a large community-based surveillance study (preprint)

Persistent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections may act as viral reservoirs that could seed future outbreaks 1-5, give rise to highly divergent lineages 6-8, and contribute to cases with post-acute Coronavirus disease 2019 (COVID-19) sequelae (Long Covid) 9,10. However, the population prevalence of persistent infections, their viral load kinetics, and evolutionary dynamics over the course of infections remain largely unknown. We identified 381 infections lasting at least 30 days, of which 54 lasted at least 60 days. These persistently infected individuals had more than 50% higher odds of self-reporting Long Covid compared to the infected controls, and we estimate that 0.09-0.5% of SARS-CoV-2 infections can become persistent and last for at least 60 days. In nearly 70% of the persistent infections we identified, there were long periods during which there were no consensus changes in virus sequences, consistent with prolonged presence of non-replicating virus. Our findings also suggest reinfections with the same major lineage are rare and that many persistent infections are characterised by relapsing viral load dynamics. Furthermore, we found a strong signal for positive selection during persistent infections, with multiple amino acid substitutions in the Spike and ORF1ab genes emerging independently in different individuals, including mutations that are lineage-defining for SARS-CoV-2 variants, at target sites for several monoclonal antibodies, and commonly found in immunocompromised patients 11-14. This work has significant implications for understanding and characterising SARS-CoV-2 infection, epidemiology, and evolution.

Viral burdens are associated with age and viral variant in a population-representative study of SARS-CoV-2 that accounts for time-since-infection related sampling bias. (preprint)

In this study, we evaluated the impact of viral variant, in addition to other variables, on within-host viral burdens, by analysing cycle threshold (Ct) values derived from nose and throat swabs, collected as part of the UK COVID-19 Infection Survey. Because viral burden distributions determined from community survey data can be biased due to the impact of variant epidemiology on the time-since-infection of samples, we developed a method to explicitly adjust observed Ct value distributions to account for the expected bias. Analysing the adjusted Ct values using partial least squares regression, we found that among unvaccinated individuals with no known prior infection, the average Ct value was 0.94 lower among Alpha variant infections, compared those with the predecessor strain, B.1.177. However, among vaccinated individuals, it was 0.34 lower among Delta variant infections, compared to those with the Alpha variant. In addition, the average Ct value decreased by 0.20 for every 10 year age increment of the infected individual. In summary, within-host viral burdens are associated with age, in addition to the interplay of vaccination status and viral variant.

Airborne Pathogen Detection in Fine Aerosol Exhaled Breath Condensates (preprint)

Rationale Exhaled breath condensate (EBC) promises a valuable, non-invasive, and easy to obtain clinical sample. However, it is not currently used diagnostically due to poor reproducibility, sample contamination, and sample loss. Objective We evaluated whether a new, hand-held EBC collector (PBM-HALE™) that separates inertially impacted large droplets (LD) before condensing the fine aerosol (FA) fraction, in distinct self-sealing containers, overcomes current limitations. Methods Sampling consistency was determined in healthy volunteers by microbial culture, 16S phylogenetics, spectrophotometry, RT-PCR, and HILIC-MS. Capture of aerosolised polystyrene beads, liposomes, virus-like particles, or pseudotyped virus was analysed by nanoparticle tracking analysis, reporter expression assays, and flow cytometry. Acute symptomatic COVID-19 case tidal FA EBC viral load was quantified by RT-qPCR. Exhaled particles were counted by laser light scattering. Measurements and Main Results Salivary amylase-free FA EBC capture was linear (R 2 =0.9992; 0.25-30 min) yielding RNA (6.03 μg/mL) containing eukaryotic 18S rRNA (RT-qPCR; p<0.001) but not human GAPDH or beta actin mRNA, and 141 non-volatile metabolites including eukaryotic cell membrane components, and cuscohygrine 3 days after cocaine abuse. Culturable aerobe viability was condensation temperature-dependent. Breath fraction-specific microbiota were stable, identifying Streptococcus enrichment in a mild dry cough case. Nebulized pseudotyped virus infectivity loss <67% depended on condensation temperature, and particle charge-driven aggregation. No SARS-CoV-2 genomes were detected in convalescent or acute COVID-19 patient tidal breath FA EBC. Conclusions High purity alveolar fraction FA EBC can reproducibly and robustly inform on contamination-free infectious agent emission sources, and be quantitatively assayed for multiple host, microbial, and lifestyle biomarker classes.

Lineage replacement and evolution captured by the United Kingdom Covid Infection Survey (preprint)

The Office for National Statistics COVID-19 Infection Survey is a large household-based surveillance study based in the United Kingdom. Here, we report on the epidemiological and evolutionary dynamics of SARS-CoV-2 determined by analysing sequenced samples collected up until 13th November 2021. We observed four distinct sweeps or partial-sweeps, by lineages B.1.177, B.1.1.7/Alpha, B.1.617.2/Delta, and finally AY.4.2, a sublineage of B.1.617.2, with each sweeping lineage having a distinct growth advantage compared to their predecessors. Evolution was characterised by steady rates of evolution and increasing diversity within lineages, but with step increases in divergence associated with each sweeping major lineage, leading to a faster overall rate of evolution and fluctuating levels of diversity. These observations highlight the value of viral sequencing integrated into community surveillance studies to monitor the viral epidemiology and evolution of SARS-CoV-2, and potentially other pathogens, particularly as routine PCR testing is phased out or in settings where large-scale sequencing is not feasible.

Applying WHO COVID-19 Workforce Estimate Tools Remotely in an African Context: A Case Report From Mali and Kenya (preprint)

Background: The COVID-19 pandemic has increased the burden on health systems, particularly in low- and middle-income countries where health systems already struggle. To meet health workforce planning needs during the pandemic, IntraHealth International used two tools created by the World Health Organization (WHO) Regional Office for Europe. The Health Workforce Estimator (HWFE) allows the estimation of the quantity of health workers needed to treat patients during a surge, and the Adaptt Surge Planning Support Tool helps to predict the timing of a surge in cases and the number of health workers and beds needed for predicted caseload. These tools were adapted to fit the African context in a rapid implementation over five weeks in one region in Mali and one region in Kenya with the objective to test the feasibility of adapting these tools, which use a Workload Indicators of Staffing Need (WISN)-inspired human resources management methodology, to obtain daily and surge projections of COVID-19 human resources for health needs.Case presentationUsing a remote team in the US and in-country teams in Mali and Kenya, IntraHealth enacted a phased plan to gather stakeholder support, collect data related to health systems and COVID-19 cases, populate data into the tools, verify modeled results with results on the ground, enact policy measures to meet projected needs, and conduct national training workshops for the ministries of health.ConclusionsThis phased implementation in Mali and Kenya demonstrated that the WISN approach applied to the Health Workforce Estimator and Adaptt tools can be readily adapted to the local context for African countries to rapidly estimate the number of health workers and beds needed to respond to the predicted COVID-19 pandemic caseload. The results may also be used to give a proxy estimate for needed health supplies—e.g., oxygen, medications, and ventilators. Challenges included accurate and timely data collection and updating data. The success of the pilot can be attributed to the adapted WHO tools, the team composition in both countries, access to human resources data, and early support of the ministries of health, with the expectation that this methodology can be applied to other country contexts.

Effect of losartan on hospitalized patients with COVID-19-induced lung injury: A randomized clinical trial (preprint)

Background SARS-CoV-2 viral entry may disrupt angiotensin II (Ang II) homeostasis in part via ACE2 downregulation, potentially contributing to COVID-19 induced lung injury. Preclinical models of viral pneumonias that utilize ACE2 demonstrate Ang II type 1 receptor (AT1R) blockade mitigates lung injury, though observational COVID-19 data addressing the effect of AT1R blockade remain mixed. Methods Multicenter, blinded, placebo-controlled randomized trial of losartan (50 mg PO twice daily for 10 days) versus placebo. Hospitalized patients with COVID-19 and a respiratory sequential organ failure assessment score of at least 1 and not already taking a renin-angiotensin-aldosterone system (RAAS) inhibitor were eligible. The primary outcome was the imputed partial pressure of oxygen to fraction of inspired oxygen (PaO 2 /FiO 2 ) ratio at 7 days. Secondary outcomes included ordinal COVID-19 severity, oxygen, ventilator, and vasopressor-free days, and mortality. Losartan pharmacokinetics (PK) and RAAS components [Ang II, angiotensin-(1–7) (Ang-(1–7)), ACE, ACE2] were measured in a subgroup of participants. Findings From April 2020 - February 2021, 205 participants were randomized, 101 to losartan and 104 to placebo. Compared to placebo, losartan did not significantly affect PaO 2 /FiO 2 ratio at 7 days [difference of -24.8 (95% -55.6 to 6.1; p=0.12)]. Losartan did not improve any secondary clinical outcome, but worsened vasopressor-free days. PK data were consistent with appropriate steady-state concentrations, but we observed no significant effect of losartan on RAAS components. Interpretation Initiation of orally administered losartan to hospitalized patients with COVID-19 and acute lung injury does not improve PaO 2 / FiO 2 ratio at 7 days. These data may have implications for ongoing clinical trials. Trial Registration Losartan for Patients With COVID-19 Requiring Hospitalization ( NCT04312009 ), https://clinicaltrials.gov/ct2/show/NCT04312009

Special Issue “Levering Sustainable Food Systems to Address Climate Change (Pandemics and Other Shocks and Hazards): Possible Transformations”

Since we first conceived of this Special Issue, “Levering Sustainable Food Systems to Address Climate Change—Possible Transformations”, COVID-19 has turned the world upside down. According to the Intergovernmental Panel on Climate Change (IPCC) [1], we have until 2030 to enact unprecedented changes to avoid the catastrophic impacts of climate change, and to adapt to unavoidable climate disruption already underway. [...]we concur with their conclusion: “Our goal must be to keep global warming below 1.5 degrees Celsius, in line with the Intergovernmental Panel on Climate Change Special Report, which recommends greenhouse gas reductions of 45 per cent below 2010 levels by 2030, and net zero emissions by 2050” [9] (p. 2). By contrast, transforming this food system model creates opportunities to help tackle climate change, improve human health, mitigate biodiversity losses (including species extinctions), renew soil health and foster more inclusive societies.

Saliva testing is accurate for early-stage and presymptomatic COVID-19 (preprint)

Background Although nasopharyngeal (NP) samples have been considered the gold standard for COVID-19 testing, variability in viral load across different anatomical sites could theoretically cause NP samples to be less sensitive than saliva or nasal samples in certain cases. Self-collected samples also have logistical advantages over NP samples, making them amenable to population-scale screening. Methods To evaluate sampling alternatives for population screening, we collected NP, saliva, and nasal samples from two cohorts with varied levels and types of symptoms. Results In a mixed cohort of 60 symptomatic and asymptomatic participants, we found that saliva had 88% concordance with NP when tested in the same testing lab (n = 41), and 68% concordance when tested in different testing labs (n = 19). In a second cohort of 20 participants hospitalized for COVID-19, saliva had 74% concordance with NP tested in the same testing lab, but detected virus in two participants that tested negative with NP on the same day. Medical record review showed that the saliva-based testing sensitivity was related to the timing of symptom onset and disease stage. Conclusions We find that no sample site will be perfectly sensitive for COVID-19 testing in all situations, and the significance of negative results will always need to be determined in the context of clinical signs and symptoms. Saliva retained high clinical sensitivity while allowing easier collection, minimizing the exposure of healthcare workers and need for personal protective equipment, and making it a viable option for population-scale testing.

A Rapid, Cost-Effective Tailed Amplicon Method for Sequencing SARS-CoV-2 (preprint)

The global COVID-19 pandemic has led to an urgent need for scalable methods for clinical diagnostics and viral tracking. Next generation sequencing technologies have enabled large-scale genomic surveillance of SARS-CoV-2 as thousands of isolates are being sequenced around the world and deposited in public data repositories. A number of methods using both short- and long-read technologies are currently being applied for SARS-CoV-2 sequencing, including amplicon approaches, metagenomic methods, and sequence capture or enrichment methods. Given the small genome size, the ability to sequence SARS-CoV-2 at scale is limited by the cost and labor associated with making sequencing libraries. Here we describe a low-cost, streamlined, all amplicon-based method for sequencing SARS-CoV-2, which bypasses costly and time-consuming library preparation steps. We benchmark this tailed amplicon method against both the ARTIC amplicon protocol and sequence capture approaches and show that an optimized tailed amplicon approach achieves comparable amplicon balance, coverage metrics, and variant calls to the ARTIC v3 approach and represents a cost-effective and highly scalable method for SARS-CoV-2 sequencing.